Takamitsu Unoki

Role of activation of PIP5Kγ661 by AP‐2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin‐mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP‐2 complex directly interacts with phosphatidylinositol 4‐phosphate 5‐kinase γ661 (PIP5Kγ661), the major PI(4,5)P2‐producing enzyme in the brain. The β2 subunit of AP‐2 was found to bind to the C‐terminal tail of PIP5Kγ661 and cause PIP5Kγ661 activation. The interaction is regulated by PIP5Kγ661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kγ661 C‐terminal region in hippocampal neurons suppresses depolarization‐dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kγ661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

NMDA Receptor-Mediated PIP5K Activation to Produce PI(4,5)P2 Is Essential for AMPA Receptor Endocytosis during LTD

NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca²⁺ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses.

Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

Phosphatidylinositol 4-Phosphate 5-Kinase Is Indispensable for Mouse Spermatogenesis

ABSTRACT The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

Polysulfide Na 2 S 4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts

Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)–Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ–S–1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN–Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides

ABSTRACT The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4‐naphthoquinone (1,4‐NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4‐NQ. In the study presented here, we determined whether 1,4‐NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4‐NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography‐tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4‐NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4‐NQ‐mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na2S2 and Na2S4, blocked 1,4‐NQ‐dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine &bgr;‐synthase (CBS) and/or cystathionine &ggr;‐lyase (CSE) enhanced these phenomena. 1,4‐NQ‐thiol adduct and 1,4‐NQ‐S‐1,4‐NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4‐NQ. The results suggest that activation of the HSP90–HSF1 signal transduction pathway mediated by 1,4‐NQ protects cells against 1,4‐NQ and that per/polysulfides can diminish the reactivity of 1,4‐NQ by forming sulfur adducts. HIGHLIGHTSHSP90 underwent covalent modification by 1,4‐NQ through Cys412 and Cys564.1,4‐NQ activated the HSP90–HSF1 signaling pathway, resulting in upregulation of HSPs.The reactive persulfide/polysulfide regulated 1,4‐NQ‐mediated HSP90–HSF1 signaling.Reactive sulfur species produced by cystathionine &ggr;‐lyase triggered sulfur adducts of 1,4‐NQ.

The Capture of Cadmium by Reactive Polysulfides Attenuates Cadmium-Induced Adaptive Responses and Hepatotoxicity

Cadmium (Cd) is an environmental electrophile that modifies protein nucleophiles, thereby modulating cellular signaling and toxicity. While reactive persulfides/polysulfides exhibit relatively high nucleophilic properties, their roles in the altered gene expression and toxicity caused by Cd remain unclear. Exposing primary mouse hepatocytes to Cd caused heat shock protein 70 (HSP70) and metallothionein (MT)-I/II to be upregulated and cytotoxicity to occur. These effects were blocked in the presence of polysulfide sodium tetrasulfide (Na2S4). Electrospray ionization mass spectrometry analysis indicated that cadmium sulfide (CdS) and cadmium thiosulfate (CdS2O3) were produced when Cd reacted with Na2S4. Authentic CdS did not cause cellular signaling responses to be activated or hepatotoxic effects, while CdS2O3 had effects similar to those of Cd. HSP70 and MT-I/II upregulation and hepatotoxicity caused by exposure to Cd were significantly enhanced by the deletion of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive persulfides/polysulfides. Deleting CSE also exacerbated Cd-mediated liver injury, whereas little hepatic damage was found when CdS or Na2S4 along with Cd was administered. Overall, the results suggest that the persulfide/polysulfide-mediated formation of sulfur adducts of Cd such as CdS rather than CdS2O3 is, at least in part, involved in decreasing the level of Cd-mediated activation of cellular signaling and toxicity.

Molecular Pathways Associated With Methylmercury-Induced Nrf2 Modulation

Methylmercury (MeHg) is a potent neurotoxin that affects particularly the developing brain. Since MeHg is a potent electrophilic agent, a wide range of intracellular effects occur in response to its exposure. Yet, the molecular mechanisms associated with MeHg-induced cell toxicity have yet to be fully understood. Activation of cell defense mechanisms in response to metal exposure, including the up-regulation of Nrf2- (nuclear factor erythroid 2-related factor 2)-related genes has been previously shown. Nrf2 is a key regulator of cellular defenses against oxidative, electrophilic and environmental stress, regulating the expression of antioxidant proteins, phase-II xenobiotic detoxifying enzymes as well phase-III xenobiotic transporters. Analogous to other electrophiles, MeHg activates Nrf2 through modification of its repressor Keap1 (Kelch-like ECH-associated protein 1). However, recent findings have also revealed that Keap1-independent signal pathways might contribute to MeHg-induced Nrf2 activation and cytoprotective responses against MeHg exposure. These include, Akt phosphorylation (Akt/GSK-3β/Fyn-mediated Nrf2 activation pathway), activation of the PTEN/Akt/CREB pathway and MAPK-induced autophagy and p62 expression. In this review, we summarize the state-of-the-art knowledge regarding Nrf2 up-regulation in response to MeHg exposure, highlighting the modulation of signaling pathways related to Nrf2 activation. The study of these mechanisms is important in evaluating MeHg toxicity in humans, and can contribute to the identification of the molecular mechanisms associated with MeHg exposure.